Dynamic evolution of ceftazidime-avibactam resistance from a single patient through the IncX3_NDM-5 plasmid transfer and blaKPC mutation.

The rapid dissemination of carbapenem-resistant Enterobacterales (CRE) especially carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a great threat to global public health. Ceftazidime-avibactam, a novel ß-lactam/ß-lactamase inhibitor combination, has been widely used due to its excellent antibacterial activity against KPC-producing K. pneumoniae. However, several resistance mechanisms have been reported since its use. Here, we conducted a series of in vitro experiments to reveal and demonstrate the dynamic evolution of ceftazidime-avibactam resistance including interspecies IncX3_NDM-5 plasmid transfer between Enterobacter cloacae and K. pneumoniae and blaKPC mutation from blaKPC-2 to blaKPC-33. Through the analysis of conjugation frequency and fitness cost, the IncX3_NDM-5 plasmid in this study showed strong transmissibility and stability in E. coli EC600 and clinical strain K. pneumoniae 5298 as recipient strain. With increasing ceftazidime-avibactam concentration, the conjugation frequency remained at 10-3-10-5, while the mutation frequency of K. pneumoniae 5298 was 10-6-10-8 at the same concentration. Further plasmid analysis (the IncX3_NDM plasmid from this study and other 658 plasmids from the NCBI database) revealed the diverse origin and genetic structure of blaNDM-5 carrying plasmids. E. coli (42.9%), China (43.9%), IncX3 (66.6%) are the most common strains, regions, and Inc types respectively. By analysing of genetic environment detected in IncX3 plasmids, the dominant structures (168/258, 65.1%) were identified: ISKox3-IS26-blaNDM-5-IS5-ISAba125-Tn3000-Tn3. In additon, several structural variations were found in the core gene structure. In conclusion, the high fitness and transmissibility of the IncX3_NDM-5 plasmids were noteworthy. More importantly, the diverse ceftazidime-avibactam resistance mechanisms including blaNDM-5 tranfer and blaKPC-2 mutation highlighted the importance of the continuous monitoring of antimicrobial susceptibility and carbapenemases subtype during ceftazidime-avibactam treatment.

Porin deficiency or plasmid copy number increase mediated carbapenem-resistant Escherichia coli resistance evolution.

This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 µg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 µg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

Occurrence and characteristics of blaOXA-181-carrying Klebsiella aerogenes from swine in China.

OBJECTIVES: Klebsiella aerogenes is a largely understudied opportunistic pathogen that can cause sepsis and lead to high mortality rates. In this study, we reported the occurrence of carbapenem-resistant blaOXA-181-carrying Klebsiella aerogenes from swine in China and elucidate their genomic characteristics. METHODS: A total of 126 samples, including 109 swine fecal swabs, 14 environmental samples, and three feed samples were collected from a pig farm in China. The samples were enriched with LB broth culture and then inoculated into MacConkey agar plates for bacterial isolation. After PCR detection of carbapenemases genes, the blaOXA-181-carrying isolates were subjected to antimicrobial susceptibility testing, and whole-genome sequence analysis. RESULTS: Four Klebsiella aerogenes isolates carrying the blaOXA-181 gene were obtained from swine faecal samples. All the 4 strains were belonged to ST438. The blaOXA-181 genes were located in IncX3-ColKP3 hybrid plasmids with the core genetic structure of IS26-ΔIS3000-ΔISEcp1-blaOXA-181-ΔlysR-ΔereA-ΔrepA-ISKpn19-tinR-qnrS1-ΔIS2-IS26, which suggests the potential for horizontal transfer and further dissemination of this resistance gene among Enterobacteriaceae and other sources. CONCLUSIONS: This study represents the first instance of OXA-181-producing K. aerogenes being identified from swine faeces in China. It is crucial to maintain continuous monitoring and ongoing attention to the detection of K. aerogenes carrying blaOXA-181 and other resistance genes in pigs.

Characterization of blaNDM-19-producing IncX3 plasmid isolated from carbapenem-resistant Escherichia coli and Klebsiellapneumoniae.

The increase in the prevalence of carbapenem-producing Enterobacterales (CPE) is a major threat, with the New Delhi metallo-ß-lactamase (NDM) enzyme-producing CPEs being one of the major causative agents of healthcare settings infections. In this study, we characterized an IncX3 plasmid harboring blaNDM-19 in Lebanon, recovered from three Escherichia coli belonging to ST167 and one Klebsiella pneumoniae belonging to ST16 isolated from a clinical setting. Plasmid analysis using PBRT, Plasmid Finder, and PlasmidSPAdes showed that all four isolates carried a conjugative 47-kb plasmid having blaNDM-19, and was designated as pLAU-NDM19. We constructed a sequence-based maximum likelihood phylogenetic tree and compared pLAU-NDM19 to other representative IncX3 plasmids carrying NDM-variants and showed that it was closely linked to NDM-19 positive IncX3 plasmid from K. pneumoniae reported in China. Our findings also revealed the route mediating resistance transmission, the IncX3 dissemination among Enterobacterales, and the NDM-19 genetic environment. We showed that mobile elements contributed to the variability of IncX3 genomic environment and highlighted that clonal dissemination in healthcare settings facilitated the spread of resistance determinants. Antimicrobial stewardship programs implemented in hospitals should be coupled with genomic surveillance to better understand the mechanisms mediating the mobilization of resistance determinants among nosocomial pathogens and their subsequent clonal dissemination.

IncX3 plasmid-mediated spread of blaNDM gene in Enterobacteriaceae among children in China.

OBJECTIVES: The blaNDM gene was prevalent among children and became the predominant cause of severe infection in infants and children. This study aimed to investigate the epidemiology and molecular characteristics of blaNDM in Enterobacteriaceae among children in China. METHODS: Carbapenem-resistant Enterobacteriaceae (CRE) were collected in the Children's Hospital of Fudan University from January 2016 to December 2022. Five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) were screened by PCR method. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. blaNDM-carrying plasmids were typed by PCR-based Incompatibility (Inc) typing method. Moreover, plasmid comparison was performed with 213 publicly available IncX3 plasmids. RESULTS: A total of 330 CRE strains were enrolled, 96.4% of which carried carbapenemase genes. blaNDM gene accounted for 64.8% (214 strains) and included four variants, including blaNDM-1 (59.8%), blaNDM-5 (39.3%), blaNDM-7 (0.5%), and blaNDM-9 (0.5%). There were no predominant MLST lineages of blaNDM carrying strains. IncX3 was the major plasmid carrying blaNDM-1 (68.0%) and blaNDM-5 (72.6%) and was dominant in blaNDM-Klebsiella penumoniae (79.8%), blaNDM-Escherichia coli (58.2%), and blaNDM-Enterobacter cloacae (61.0%), respectively. Most (79.0%) clinical IncX3 plasmids in the world carried blaNDM, and the prevalence of blaNDM in IncX3 plasmids was more common in China (95.8%) than other countries (58.1%, P <0.01). CONCLUSION: blaNDM is highly prevalent in CRE among children in China. The spread of blaNDM was mainly mediated by IncX3 plasmids. Surveillance and infection control on the spread of blaNDM among children are important.

Transmission of blaNDM in Enterobacteriaceae among animals, food and human.

Despite carbapenems not being used in animals, carbapenem-resistant Enterobacterales (CRE), particularly New Delhi metallo-ß-lactamase-producing CRE (NDM-CRE), are prevalent in livestock. Concurrently, the incidence of human infections caused by NDM-CRE is rising, particularly in children. Although a positive association between livestock production and human NDM-CRE infections at the national level was identified, the evidence of direct transmission of NDM originating from livestock to humans remains largely unknown. Here, we conducted a cross-sectional study in Chengdu, Sichuan Province, to examine the prevalence of NDM-CRE in chickens and pigs along the breeding-slaughtering-retail chains, in pork in cafeterias of schools, and in colonizations and infections from children's hospital and examined the correlation of NDM-CRE among animals, foods and humans. Overall, the blaNDM increases gradually along the chicken and pig breeding (4.70%/2.0%) -slaughtering (7.60%/22.40%) -retail (65.56%/34.26%) chains. The slaughterhouse has become a hotspot for cross-contamination and amplifier of blaNDM. Notably, 63.11% of pork from the school cafeteria was positive for blaNDM. The prevalence of blaNDM in intestinal and infection samples from children's hospitals was 21.68% and 19.80%, respectively. whole genome sequencing (WGS) analysis revealed the sporadic, not large-scale, clonal spread of NDM-CRE along the chicken and pig breeding-slaughtering-retail chain, with further spreading via IncX3-blaNDM plasmid within each stage of whole chains. Clonal transmission of NDM-CRE is predominant in children's hospitals. The IncX3-blaNDM plasmid was highly prevalent among animals and humans and accounted for 57.7% of Escherichia coli and 91.3% of Klebsiella pneumoniae. Attention should be directed towards the IncX3 plasmid to control the transmission of blaNDM between animals and humans.

Genomic characterisation of blaNDM-5-IncX3 plasmid in an ST4 Klebsiella aerogenes clinical isolate.

OBJECTIVES: The dissemination of New Delhi metallo-ß-lactamase-5 (NDM-5) among various species of Enterobacterales has attracted serious global attention. Here, we characterise the genomic characterisation of blaNDM-5-IncX3 plasmid (pNDM-KA3) in an ST4 Klebsiella aerogenes (KA3) strain isolated from a neonate with pneumonia. METHODS: Antimicrobial susceptibility and multilocus sequence typing was performed for the KA3. The plasmid conjugation assay and plasmid stability of the KA3 (pNDM-KA3) were also analysed. The pNDM-KA3 plasmid was further analysed by whole-genome sequencing and comparative analysis to determine the genetic environment of blaNDM-5. RESULTS: The KA3 strain belongs to ST4 and shows high resistance to ß-lactam antibiotics, including carbapenems, but is susceptible to ciprofloxacin, amikacin, tigecycline, and colistin. The pNDM-KA3 was successfully transferred to the recipient E. coli J53 and showed strong stability in K. aerogenes. Genomic sequencing revealed that the pNDM-KA3 plasmid was assigned to plasmid incompatibility group X3 with 43367 bp, and a conserved structure sequence of △IS3000-△ISAba125-IS5-blaNDM-5-bleMBL- trpF-dsbC-IS26 was detected upstream and downstream of the blaNDM-5 gene. Further analysis revealed that insertion sequences mediated the dissemination of blaNDM-5 from other species of Enterobacterales. The pNDM-KA3 showed high similarity to blaNDM-5-harbouring plasmids in other species of Enterobacterales, with these plasmids carrying genes for replication (repB), partitioning (parA and parB), stability (hns), and conjugative transfer (virB and virD). CONCLUSIONS: Continued monitoring for the dissemination of blaNDM-5 among uncommon Enterobacterales species should be further reinforced.

Clone dissemination of IncX3 plasmid carrying blaNDM-1 in ST76 carbapenem resistance Klebsiella pneumoniae and bactericidal efficiency of aztreonam combined with avibactam in vitro and in vivo.

OBJECTIVES: The rapid spread of the New Delhi Metal-ß-lactamase-1 (NDM-1) gene in Klebsiella pneumoniae poses a substantial challenge to pediatric therapeutic care. Here, we aimed to characterise the IncX3-type plasmid carrying the blaNDM-1 gene in ST76 carbapenem resistance K. pneumoniae (CRKP) strains and assess the in vitro and in vivo bactericidal efficacy of Aztreonam (ATM) combined with Avibactam (AVI) (ATM+AVI) against CRKP. METHODS: The broth microdilution method and PCR were used to detect antimicrobial susceptibility and antibiotic resistance genes. Genetic relatedness was determined using Pulsed-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). The plasmid conjugation assay was used to verify the transmissibility of drug-resistant plasmids. Whole-Genome Sequencing (WGS) was employed to elucidate the genomic attributes of the genes. The Fractional Inhibitory Concentration (FIC) was calculated based on the checkerboard titration assay to determine the antimicrobial effect of ATM+AVI. The time-kill curve assay and a mouse anti-infection model were used to investigate the in vitro and in vivo bactericidal efficiency of ATM+AVI. RESULTS: Seven blaNDM-1-producing strains were found to be highly resistant to carbapenems, and they all belonged to the same sequence type (ST76) and were classified into the same PFGE clusters with an 89.1% similarity. The conjugation assay showed that the blaNDM-1-carrying plasmid was successfully transferred to Escherichia coli 600, resulting in transconjugants with carbapenem antibiotic resistance. A 54-kb IncX3 plasmid (pNDM-XZA88) carried the blaNDM-1 gene located on a Tn125 transposon-like element structure, demonstrating the transferability of resistance genes. Genome comparative analysis revealed that pNDM-XZA88 was highly similar to pCQ17 × 3 and pRor-30818cz and had relatively conserved backbones and variable accessory regions compared to the other four plasmids (pC39-334 kb, pNDM-1-DY1928, pNDM-K725, and pNDM-Z244). The checkerboard titration and time-kill curve assays revealed that the ATM+AVI combination therapy exerted significant bactericidal efficacy against the blaNDM-1-producing strains in vitro. The ATM+AVI combination also significantly reduced the bacterial burden in a mouse infection model constructed using the blaNDM-1-producing K. pneumoniae. CONCLUSION: This study demonstrated the clone dissemination of blaNDM-1-harboring IncX3 plasmids among the ST76 K. pneumoniae isolated from pediatric patients. Therefore, more attention should be paid to preventing this high-risk clone from harming pediatric patients. Moreover, we deduced that the ATM+AVI combination therapy is an effective strategy for treating blaNDM-1-producing K. pneumoniae.

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid.

OBJECTIVES: Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. METHODS: The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). RESULTS: CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. CONCLUSION: In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Genetic and phenotypic characterizations of IncX3 plasmids harboring bla NDM-5 and bla NDM-16b in Japan.

IMPORTANCE: IncX3 plasmids harboring bla NDM-5 play a major role in the spread of carbapenem resistance in Asia, particularly in China, in clinical and environmental settings. In this study, we present that Enterobacterales isolates carrying IncX3 plasmids harboring bla NDM-5 have been disseminated in Japan, where their identification was previously rare. In addition, bla NDM-16b, a single-nucleotide variant of bla NDM-5, was found to be carried by an identical IncX3 plasmid. A comparative sequence analysis revealed that the bla NDM-16b gene emerged from a single nucleotide substitution on an IncX3 plasmid harboring bla NDM-5. The bla NDM-16b gene did not confer elevated carbapenem resistance compared to bla NDM-5 in our assay using transformants carrying the plasmid harboring either of these genes, although the A233V substitution was reported to confer stability to the enzyme in ion-depleted conditions. Nevertheless, vigilance regarding the emergence of novel variants is required.

Whole genome sequence of carbapenem-resistant hypermucoviscous Klebsiella pneumoniae K2-ST375 with blaNDM-harbouring conjugative IncX3 and pLVPK-like virulence plasmids from a patient in China.

OBJECTIVES: Carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP) poses unprecedented public health challenges. However, genomic information regarding the CR-HMKP K2-ST375 strain is scarce. The aim of this study was to characterize the whole genome sequence of the CR-HMKP K2-ST375 strain Kp0179 isolated from a male patient in China. METHODS: The whole genome of Kp0179 was sequenced using the DNBSEQ and Pacific Biosciences RSII platforms. The capsular serotype, multilocus sequence typing (MLST), antimicrobial resistance genes, and virulence factors were determined using available databases and bioinformatics tools. Conjugation experiments were performed using rifampicin-resistant Escherichia coli C600 as the recipient. RESULTS: The Kp0179 strain with hypermucoviscous phenotype was resistant to almost all ß-lactams, including ertapenem and imipenem. Whole genome sequencing revealed that Kp0179 belonged to K2-ST375 and contained blaNDM-IncX3 and a virulence plasmid ca. 121 kb. Kp0179 contained 5146 coding genes, 88 tRNAs, 25 rRNAs and 38 non-coding RNA genes. Among the six acquired antibiotic resistance genes, blaSHV-99, fosA, oqxAB were located on the chromosome, whereas blaNDM-1, qnrS1 and blaSHV-12 were located on the conjugative plasmid pNDM-Kp0179 (IncX3 type). Virulence gene analysis indicated that pLVPK-Kp0179 carried multiple virulence-encoding genes, such as iroBCDN, iucABCDiutA, rmpA and rmpA2. In addition to carrying a virulence plasmid, capsule formation (kvgA) and the type 3 fimbriae operon (mrkABCDFHIJ) were located on the chromosome of Kp0179. CONCLUSION: To our knowledge, this is the first report of a CR-HMKP K2-ST375 strain with a blaNDM-harboured conjugative IncX3 plasmid and a pLVPK-like virulence plasmid from a patient in China. Therefore, the spread of CR-HMKP K2-ST375 isolates in China should be closely monitored.

Molecular characterization and comparison of bla NDM-1-carrying and bla NDM-5-harboring IncX3-type plasmids in carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP), which harbors the bla NDM plasmid, has been reported extensively and is considered a global threat clinically. However, characterization and comparisons of bla NDM-1-carrying and bla NDM-5-harboring IncX3-type plasmids in CRKP are lacking. Here, we systematically compared the differences in the characteristics, genetic backgrounds, transferability, and fitness costs between bla NDM-1-carrying and bla NDM-5-carrying plasmids in K. pneumoniae isolates. Fifteen NDM-producing CRKP isolates were recovered from 1376 CRKP isolates between 2019 and 2021, of which 4 were positive for bla NDM-1 and 11 were positive for bla NDM-5. All strains were highly resistant to carbapenem but remained susceptible to tigecycline and colistin. Core-genome-based phylogenetic analyses revealed that these strains were not clonally related. Whole-genome sequencing showed that bla NDM-1 and bla NDM-5 were located on ~54 kb and ~46 kb IncX3-type plasmids, respectively. The backbone, genetic context, and fitness cost of the bla NDM-1-bearing plasmid were highly similar to those of the bla NDM-5-carrying plasmid, but the transferability of the bla NDM-1-positive plasmid was greater than that of the bla NDM-5-positive plasmid. In conclusion, the transmission of bla NDM-1 or bla NDM-5 is mainly disseminated by plasmids rather than clonal spread. The high transfer frequency of the IncX3 plasmid facilitates the prevalence and dissemination of NDM-KP among Enterobacteriaceae. IMPORTANCE The emergence of NDM-producing Klebsiella pneumoniae is a severe challenge to public health. The widespread presence of bla NDM-1 and bla NDM-5 in Enterobacteriaceae has aroused broad concern. In this study, we performed molecular characterization of bla NDM-1-carrying and bla NDM-5-harboring IncX3-type plasmids in carbapenem-resistant Klebsiella pneumoniae (CRKP) and compared their phenotypes between strains with different bla NDM subtype. Our findings highlight the importance of IncX3-type plasmids in the transfer of the bla NDM-1 and bla NDM-5 genes and demonstrate that the bla NDM-1 plasmid possesses higher transfer ability. These data will provide important insights into carbapenem resistance gene transfer via plasmids and their further spread in clinical settings.

Emergence of OXA-484-Producing Klebsiella variicola in China.

Purpose: The frequent and inappropriate use of antibiotics has caused a dramatic rise in the number, species, and degree of multi-drug resistant bacteria, making them more prevalent and difficult to treat. In this context, the aim of the present study was to characterize the OXA-484-producing strains isolated from a perianal swab of a patient by using whole-genome analysis. Patients and Methods: In this study, carbapenemase-producing Klebsiella variicola was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), average nucleotide identity (ANI) and PCR. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were utilized to characterize the plasmid profiles of K. variicola 4717. In particular, WGS was performed to obtain genomic information on this clinical isolate, and assemble all the plasmids of the bla OXA-484-harboring strain. Results: The antimicrobial susceptibility pattern of K. variicola 4717 revealed that it was resistant to a range of antibiotics, including aztreonam, imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, levofloxacin, ciprofloxacin, piperacillin-tazobactam, methylene-sulfamer oxazole, amoxicillin-clavulanic acid, cefepime, and tigecycline. Its susceptibility to chloromycin was intermediate, while it was still susceptible to amikacin, gentamicin, fosfomycin, and polymyxin B. The presence of two companion plasmids, p4717_1 and p4717_2, together with a plasmid carrying the bla OXA-484 gene was observed. An in-depth investigation of p4717-OXA-484 uncovered that it is an IncX3-type plasmid and shares a similar segment encoded by IS26. Given the similar genetic background, it was conceivable that bla OXA-484 could have developed from bla OXA-181 through a series of mutations. Conclusion: Herein, we described the first genome sequence of K. variicola strain harbouring the class D ß-actamase bla OXA-484 in an Inc-X3-type plasmid. Our work also uncovered the genetic characterization of K. variicola 4717 and the importance of initiating antimicrobial detection promptly.

First Report of OXA-181-Producing Enterobacterales Isolates in Latin America.

We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the ß-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

Emergence of blaSHV-12 and qnrS1 encoded on IncX3 plasmids: Changing epidemiology of extended-spectrum ß-lactamases among Enterobacterales isolated from broilers.

OBJECTIVES: The occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in broilers represents a risk to public health because of the possibility of transmission of ESBL producers and/or blaESBL genes via the food chain or within settings where human-animal interfaces exist. METHODS: This study assessed the occurrence of ESBL producers among faecal samples of broilers at slaughter. Isolates were characterised by multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing. RESULTS: The flock prevalence, determined by sampling crates of 100 poultry flocks, was 21%. The predominant blaESBL gene was blaSHV-12, identified in 92% of the isolates. A variety of Escherichia coli and Klebsiella pneumoniae sequence types (STs) were identified, including extraintestinal pathogenic E. coli ST38, avian pathogenic E. coli ST10, ST93, ST117, and ST155, and nosocomial outbreak clone K. pneumoniae ST20. Whole-genome sequencing was used to characterise a subset of 15 isolates, including 6 E. coli, 4 K. pneumoniae, 1 Klebsiella grimontii, 1 Klebsiella michiganensis, 1 Klebsiella variicola, and 1 Atlantibacter subterranea. Fourteen isolates carried identical or closely related 46338-54929 bp IncX3 plasmids encoding blaSHV-12 and qnrS1. One E. coli isolate carried a 46338 bp IncX3 plasmid, which was integrated chromosomally into ydbD. CONCLUSIONS: The blaSHV-12 gene has replaced the previously predominant blaCTX-M-1 in ESBL-producing Enterobacterales from broilers in Switzerland. Broilers may play a role in the dissemination of blaSHV-12 and qnrS1 associated with epidemic IncX3 plasmids, representing a risk to human and animal health.

Adaptive Evolution Compensated for the Plasmid Fitness Costs Brought by Specific Genetic Conflicts.

New Delhi metallo-ß-lactamase (NDM)-carrying IncX3 plasmids is important in the transmission of carbapenem resistance in Escherichia coli. Fitness costs related to plasmid carriage are expected to limit gene exchange; however, the causes of these fitness costs are poorly understood. Compensatory mutations are believed to ameliorate plasmid fitness costs and enable the plasmid's wide spread, suggesting that such costs are caused by specific plasmid-host genetic conflicts. By combining conjugation tests and experimental evolution with comparative genetic analysis, we showed here that the fitness costs related to ndm/IncX3 plasmids in E. coli C600 are caused by co-mutations of multiple host chromosomal genes related to sugar metabolism and cell membrane function. Adaptive evolution revealed that mutations in genes associated with oxidative stress, nucleotide and short-chain fatty acid metabolism, and cell membranes ameliorated the costs associated with plasmid carriage. Specific genetic conflicts associated with the ndm/IncX3 plasmid in E. coli C600 involve metabolism and cell-membrane-related genes, which could be ameliorated by compensatory mutations. Collectively, our findings could explain the wide spread of IncX3 plasmids in bacterial genomes, despite their potential cost.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter strains in China: a multicenter genomic study.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter (CR-Ent) species remain unclear in China. In this study, we performed a genomic study on 92 isolates from Enterobacter-caused infections from a multicenter study in China. Whole genome sequencing (WGS) was used to determine the genome sequence of 92 non-duplicated CR-Ent strains collected from multiple tertiary health centres. The precise species of Enterobacter strains were identified by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH). Molecular features of high-risk CR-Ent sequence type (ST) lineages and carbapenemase-encoding plasmids were determined. The result revealed that the most common human-source CR-Ent species in China was E. xiangfangensis (66/92, 71.93%), and the proportion of carbapenemase-producing Enterobacter (CP-Ent) in CR-Ent was high (72/92, 78.26%) in comparison to other global regions. Furthermore, ST171 and ST116 E. xiangfangensis were the major lineages of CP-Ent strains, and ST171 E. xiangfangensis was more likely to cause infections in older patients. Genomic analysis also highlighted the likelihood of intra-hospital/inter-hospital clonal transmission of ST171 and ST116 E. xiangfangensis. In addition, the blaNDM-harbouring IncX3-type plasmid was identified as the prevalent carbapenemase-encoding plasmid carried by CR-Ent strains, and was experimentally confirmed to be able to self-transfer with high frequency. This study detailed the genomic and clinical characteristics of CR-Ent in China in the form of multicenter for the first time. The high risk of carbapenemase-producing ST171 and ST116 E. xiangfangensis, and the blaNDM-harbouring IncX3-type plasmid were detected and emphasized.

Klebsiella Species and Enterobacter cloacae Isolates Harboring blaOXA-181 and blaOXA-48: Resistome, Fitness Cost, and Plasmid Stability.

IncX3 and IncL plasmids have been named as catalysts advancing dissemination of blaOXA-181 and blaOXA-48 genes. However, their impact on the performance of host cells is vastly understudied. Genetic characteristics of blaOXA-48- and blaOXA-181-containing Klebsiella pneumoniae (EFN299), Klebsiella quasipneumoniae (EFN262), and Enterobacter cloacae (EFN743) isolated from clinical samples in a Ghanaian hospital were investigated by whole-genome sequencing. Transfer of plasmids by conjugation and electroporation, plasmid stability, fitness cost, and genetic context of blaOXA-48, blaOXA-181, and blaDHA-1 were assessed. blaOXA-181 was carried on two IncX3 plasmids, an intact 51.5-kb IncX3 plasmid (p262-OXA-181) and a 45.3-kb IncX3 plasmid (p743-OXA-181) without replication protein sequence. The fluoroquinolone-resistant gene qnrS1 region was also excised, and unlike in p262-OXA-181, the blaOXA-181 drug-resistant region was not found on a composite transposon. blaOXA-48 was carried on a 74.6-kb conjugative IncL plasmid with unknown ~10.9-kb sequence insertion. This IncL plasmid proved to be highly transferable, with a conjugation efficiency of 1.8 × 10-2. blaDHA-1 was present on an untypeable 22.2 kb genetic structure. Plasmid stability test revealed plasmid loss rate between 4.3% and 12.4%. The results also demonstrated that carriage of IncX3-blaOXA-181 or IncL-blaOXA-48 plasmids was not associated with any fitness defect, but rather an enhanced competitive ability of host cells. This study underscores the significant contribution of IncX3 and IncL plasmids in the dissemination of resistance genes and their efficient transfer calls for regular monitoring to control the expansion of resistant strains. IMPORTANCE The growing rate of antibiotic resistance is an important global health threat. This threat is exacerbated by the lack of safe and potent alternatives to carbapenems in addition to the slow developmental process of newer and effective antibiotics. Infections by carbapenem-resistant Gram-negative bacteria are becoming almost untreatable, leading to poor clinical outcomes and high mortality rates. OXA-48-like carbapenemases are one of the most widespread carbapenemases accounting for resistance among Enterobacteriaecae. We characterized OXA-48- and OXA-181-producing Enterobacteriaecae to gain insights into the genetic basis and mechanism of resistance to carbapenems. Findings from the study showed that the genes encoding these enzymes were carried on highly transmissible plasmids, one of which had sequences absent in other similar plasmids. This implies that mobile genetic elements are important players in the dissemination of resistance genes. Further characterization of this plasmid is warranted to determine the role of this sequence in the spread of resistance genes.

Global prevalence, characteristics, and future prospects of IncX3 plasmids: A review.

IncX3 plasmids are narrow host range plasmids mostly found in Enterobacteriaceae with great conjugation ability, high stability, no fitness cost, and the ability to improve biofilm formation in their bacterial hosts. IncX3 plasmids have spread swiftly, primarily in several nations and among different species over the last 10 years. blaNDM , blaKPC , and blaOXA-181 are the carbapenemase genes carried by IncX3 plasmids. Among them, blaNDM is often located on the IncX3 plasmid, which is deemed as the primary vehicle of blaNDM transmission. Isolates harboring IncX3 plasmids are found in nations all over the world from human, animal, and environmental sources. Cointegrate plasmids related to IncX3 have recently been discovered to increase the antibiotic resistance spectrum and potentially broaden the host range of plasmids, restricting the use of antibiotics in the clinic. There are, however, few reviews based on the physiological and epidemiological properties of IncX3 plasmid, as well as studies on the plasmid itself. Hence, we conducted a retrospective literature review to summarize the characteristics of IncX3 plasmids aiming to provide a theoretical basis for controlling the global prevalence of IncX3 plasmids and directions for further research on the functions of the related genes on the IncX3 plasmid.

Case Report of bla NDM-7-Harboring IncX3 Plasmid in ST196 Klebsiella quasipneumoniae in China.

Klebsiella quasipneumoniae isolate SBH035 was recovered from a patient in Jiangsu Province, China. The isolate showed resistance to ampicillin, cefazolin, cefotaxime, meropenem, ceftazidime-avibactam, and fosfomycin. The carbapenemase-encoding gene bla NDM-7 was identified, and whole genome sequencing analysis indicated that bla NDM-7 was located in an IncX3 plasmid with a conserved structure of IS26-ΔcutA-tat-trpF-ble MBL -bla NDM-7-ISAba125-IS3000-ΔTn2. To date, this is the first identification of a bla NDM-7-harboring IncX3 plasmid in ST196 K. quasipneumoniae from a patient in China. Greater attention to controlling the dissemination of IncX3 plasmids is needed owing to potential horizontal transfer via mobile genetic elements.